The present application is directed to reagents and methods of capillary electrophoresis, which are suitable for measuring nonenzymatically glycosylated proteins in clinical specimens. These materials and methods are particularly useful for monitoring glycemic control in diabetics.
Assays for glycated hemoglobin have been performed in clinical laboratories for well over a decade. Analysis of normal hemoglobin, Hb A, has shown that it contains a number of minor hemoglobin species. These minor species, which have been designated Hb A1a, Hb A1b, and Hb A1c, are referred to as glycated hemoglobins or glycohemoglobins. They are formed by condensation of an amino group of hemoglobin with an aldehyde group of a reducing sugar. For Hb A1c, glycated hemoglobin is formed by the condensation of the N-terminal valine of the hemoglobin beta chain with glucose to form an unstable Schiff base or aldimine (also known as pre-A1c), which then undergoes an Amadori rearrangement to form a stable ketoamine.
The formation of glycated hemoglobin is nonenzymatic. It occurs over the lifespan of the red blood cell, which is about 120 days under normal conditions. The amount of glycated hemoglobin formed is also proportional to the concentration of glucose in the blood. Consequently, the concentration of Hb A1c in the blood is related to the time-averaged glucose concentration over the two or three month period prior to measurement. This value provides a way of measuring control of diabetes, where the results are not affected by short-term fluctuations in plasma glucose levels. Therefore, measurement of glycohemoglobins can complement other more traditional methods of assessing control of diabetes. For example, measurement of glycohemoglobins can be used to verify patient compliance, where self administered urine or blood glucose records may be falsified or blood glucose levels vary markedly throughout the day or from day to day. Other applications include new patients with known or suspected diabetes in whom there is no previous record of blood glucose concentration or during pregnancy when close control of diabetes is especially important.
Currently available methods for the determination of glycohemoglobin can be divided into two different categories. The first category includes methods, such as colorimetry, affinity chromatography, and immunoassay, that separate minor hemoglobin components based on the structural characteristics of sugar moieties on hemoglobin. The second category, which includes ion exchange chromatography, high-performance liquid chromatography, electrophoresis, and isoelectric focusing, separates hemoglobin components based on charge differences between glycosylated and nonglycosylated proteins. A comparison of these methods has been reported (Ralph E. Bernstein, "Nonenzymatically Glycosylated Proteins," Advances in Clinical Chemistry, Vol. 26, 1-78 (1987)).
A colorimetric method has been devised based on the observation that Hb A1c, when subject to mild acid hydrolysis, releases 5-hydroxymethylfurfural (5-HMF). This test has proven difficult to standardize because the yield of 5-HMF from Hb A1c is only about 30%. In order to provide reliable results, reaction conditions must be carefully controlled for inter alia temperature, pressure, and time. A further drawback is the use of oxalic acid and thiobarbituric acids, which are potentially toxic chemicals. Therefore, this method is unsuitable for routine clinical analysis, particularly when rapid results are needed.
Separation of nonglycated hemoglobin from glycated hemoglobin by affinity chromatography capitalizes on the ability of boronates to form complexes with sugars. A suitable affinity column is prepared from a gel containing immobilized m-aminophenylboronic acid on cross-linked, beaded agarose. The boronic acid reacts with the cis-diol groups of glucose bound to hemoglobin to form a reversible 5-membered ring complex, thus selectively binding the glycated hemoglobin to the affinity column. The nonglycated hemoglobin passes through the column. The glycated hemoglobin is then dissociated from the complex by sorbitol. Although this method is less susceptible to variations in temperature or ionic conditions than other methods, such as colorimetry and ion-exchange chromatography, the affinity columns must be protected from sunlight and can only be reused a limited number of times before they must be discarded.
Antibody against Hb A1c can be prepared and used as the basis for a radioimmunoassay. However, such a radioimmunoassay, like radioimmunoassays in general, brings with it the problems of the disposal of reagents and the short shelf life of reagents due to degradation caused by radioactive labeling, with consequent loss of specific reactivity. Thus radioimmunoassay, though capable of accuracy, cannot generally be used for routine determinations of Hb A1c.
Ion exchange chromatography can be carried out using resins containing weakly acidic cation exchange or negatively charged carboxymethylcellulose resin. This procedure is time consuming and requires rigid control of temperatures of the reagents and the columns as well as the pH and the ionic strength. In practice, this means that the methods are usable only by highly skilled personnel and are not well suited to routine clinical determinations.
High performance liquid chromatography (HPLC) is a highly instrumental technique that is more costly initially, but is rapid, requires small samples, and can be automated. HPLC usually has great sensitivity and excellent reproducibility for the isolation of glycated hemoglobins. However, the accurate quantitation of Hb A1c by HPLC can be hampered when fetal hemoglobin, HbF, is present in elevated amounts.
Electrophoretic assays for Hb A1c or other glycoslyated proteins have given mixed results. Although Hb A1c is more negatively charged than Hb A.sub.0, their isoelectric points differ by only 0.01 pH units. Consequently, modified methods like "mobile affinity electrophoresis", have been developed to magnify minor charge differences. Mobile affinity electrophoresis adds dextran sulfate to the buffers. This results in binding of sulfate groups to the nonglycated hemoglobins and acceleration of their electrophoretic mobilities. In another modified electrophoretic method, the fixed negative charge of the agar gel matrix can result in differential interaction with Hb A.sub.o and Hb A1c causing the separation of hemoglobins due to electroendoosmotic effects. Compared to mobile affinity electrophoresis, the agar gel method has given adequate, though less reproducible, results. A third electrophoretic method, Isoelectric focusing (IEF), provides definitive separation of glycated hemoglobins, unmatched by other charge dependent methods. However, the stability and reproducibility of the pH gradients required for this technique are tenuous at best. All electrophoretic methods developed to date require the skillful execution of numerous time consuming steps.
Each of these methods present problems of safety, accuracy, reproducibility, or efficiency. What is needed to overcome these problems is an assay that accurately and precisely distinguishes interaction with Hb A.sub.o and Hb A.sub.1c causing the separation of hemoglobins due to electroendoosmotic effects. Ideally, the assay is also rapid, efficient, uses inexpensive nontoxic reagents, has reusable equipment, and is amenable to automation for autosampling, on-line detection, and data acquisition.